Natural killer (NK) cells are a major lineage of human lymphocytes with vital functions in innate immunity, adaptive immunity and hematopoietic cell transplantation. These functions are mediated by the diverse interactions of highly polymorphic HLA-A, -B, and -C molecules with equally polymorphic killer immunoglobulin- like receptors (KIR). They are complemented by the conserved interactions of HLA-E with CD94:NKG2 heterodimers. The genes encoding KIR receptors and HLA ligands segregate independently, thereby generating unique and diverse genotypes within families and populations. Phenotypically, this produces functionally distinct NK-cell repertoires, a natural variation that has profound effects on susceptibility to infection and autoimmunity, on reproductive success, and the outcome of HCT. In SA1 we will extend retrospective statistical analyses of KIR and HLA variation in HCT to the highest levels of allele-level resolution and precision. We have developed a novel target capture and next-generation sequencing methodology with compatible bioinformatics tools. These will be used to determine complete KIR haplotype sequences and define allelic and allotypic variation throughout the KIR locus. In SA2 we will perform high-resolution analysis of immune reconstitution following HCT. Three cohorts will be studied -- HLA-matched, HLA-mismatched, and HLA-haploidentical transplants ? by comparing the mature NK cells of the donor to the reconstituting NK cells in the transplant recipient. Longitudinal differences in immune phenotype will be assessed and compared. Functional capacity of the NK cells will be tested by challenge with target cells expressing self, altered-self, non-self and missing-self HLA class I. Early, intermediate and late stages in immune system reconstitution will be evaluated. We will assess the reconstituted NK-cell response to exogenous stimuli (the CD16 directed- BiKEs and IL-15/IL-15R?-Fc superagonist complexes also studied in Projects 2 and 3) at the different stages. All analyses will consider KIR and HLA genotypes and CMV status. NK cells mature to acquire functional immunity through a process called education. SA3 of this project will analyze mature, educated NK cells and determine their functional capacity to recognize and respond to target cells expressing self, non-self, altered- self, and missing-self HLA class I. To achieve this goal, we will develop a high-throughput assay to assess NK- cell responses to an extensive cell panel, in which each member cell expresses a different HLA class I allotype. From these data, predictive rules for education, based on the KIR and HLA genotype and CMV status of individuals, will be defined. Mass cytometry will be used to analyze NK cells and other lymphocyte subpopulations for individuals with selected combinations of KIR, HLA and CMV status. The results from this project will exponentially increase knowledge of the genetic factors influencing the outcome of HCT. Our long- term goal is to obtain complete immunogenetic and biological understanding of the parameters affecting NK cell education. This knowledge should inform a wide range of studies aimed at improving human health.